Nonmuscle (nm) myosin is an essential part of the cytoskeleton, a complex intracellular network that orchestrates such cell functions as cytokinesis, motility, and muscle contraction. Recent work has demonstrated that dictyostelium lacking myosin heavy chain (mhc) are viable, motile, and phagocytic, although they demonstrate markedly abnormal cytokinesis. In dividing mammalian cells, nm myosin localizes to the cleavage furrow during cytokinesis; labeled myosin light chains microinjected into living cells localize in the cleavage furrow, and become reincorporated into stress fibers at the completion of cytokinesis. Nm myosin may also be necessary for sarcomere assembly, because the cytoskeleton must act as a scaffolding for synthesis of the sarcomeric contractile proteins to occur by cotranslational assembly. Exciting advances in gene targeting now make it possible to target nonselectable genes by homologous recombination. Preliminary data suggests that the nm mhc gene is encoded by one or a few genes making such gene targeting feasible. The primary goal of this project is to complete the cloning and characterization of the nm mhc gene, in order to begin gene disruption experiments by homologous recombination. By disrupting the nm mhc in mouse non and muscle cells, we should be able to elucidate the role of nm mhc in cytokinesis, cell motility and sarcomere assembly. Further studies will focus on identifying those gene domains that confer specific functions to the nm mhc protein in both muscle and nonmuscle.